Prognostic role of the updated Kiel classification in canine high‐grade T‐cell lymphomas

Abstract Background The aim of this study was to determine and describe the prognostic role of the morphological subtype determined according to the updated Kiel classification in dogs with high‐grade T‐cell lymphomas (HGTCLs) depending on the treatment applied. Objectives The HGTCLs were classified into three subtypes according to the updated Kiel classification: pleomorphic mixed (PM), lymphoblastic lymphoma/acute lymphoblastic leukaemia and plasmacytoid (P). The treatment was divided into a palliative therapy (PlT) group and a chemotherapy (ChT) group. Methods The study was conducted between 2009 and 2017, and it enrolled 58 dogs in which cytomorphological and immunocytochemistry diagnoses were HGTCL. Results Overall survival (OS) was significantly longer in the ChT group (median OS—4 months, interquartile range [IQR] from 2 to 8 months) than in the PlT group (median OS—6 weeks, IQR from 1 week to 3 months). In the PlT group, PM subtype and glucocorticosteroids (GCSs) treatment proved significantly and independently linked to longer OS and approximately three‐fold lower risk of death during the study period (adjusted hazard ratio [HRadj] = 0.26, confidence interval [CI] 95%: 0.08–0.81; p = 0.020 and HRadj = 0.30, CI 95%: 0.11–0.77; p = 0.013, respectively), although due to small group size, precision of estimations was poor (wide CI 95%). In the ChT group, >7 days elapsing between diagnosis and the beginning of chemotherapy and GCS treatment prior to chemotherapy were significantly associated with lower chance of complete remission (CR; p = 0.034 for both); GCS treatment prior to chemotherapy was significantly associated with shorter OS (p = 0.016); chemotherapy based on the modified CHOP protocol was significantly associated with higher chance of CR (p = 0.034) and longer OS (p = 0.039); and CR was significantly linked to longer OS (p = 0.001). Clinical significance The morphological subtype of HGTCL has some prognostic value in dogs treated palliatively (with PM subtype associated with longer OS than P subtype); however, this effect is no longer visible when a dog is treated with chemotherapy.


INTRODUCTION
Canine T-cell lymphomas (TCLs) are a heterogeneous group of neoplasms, with different biologic behaviour and response to treatment.
However, TCL may be classified into high-grade TCL (HGTCL) and lowgrade TCL (e.g.T-zone lymphoma), with the latter having an indolent course (Seelig et al., 2014).Therefore, using the immunophenotype as the only prognostic factor does not seem to be sufficient (Ponce et al., 2004;Valli et al., 2006).According to the Revised European-American Classification of Lymphoid Neoplasms and World Health Organisation (WHO), the current classification of canine lymphomas is based on cytologic/histologic parameters (morphotype, i.e., cell morphology and lymph node tumour architecture), immunophenotype (B or T), cytogenetic profile, clinical and epidemiological data (Campo et al., 1998;Harris et al., 1994).The morphologic classification based on the updated Kiel classification adapted to the canine species has generally proven to have some prognostic significance in canine lymphomas (Ponce et al., 2004;Sayag et al., 2018).In this study, we decided to focus on the prognostic role of the morphological subtype determined according to the updated Kiel classification in dogs with HGTCL depending on the treatment applied.

Study population
The study was conducted between 2009 and 2017, and it enrolled 58 dogs in which cytomorphological diagnosis of the high-grade lymphoma was made and identified using the immunocytochemistry as HGTCL twice by the same clinical cytologist (the period between first examination and re-examination of slides was 1-2 months).
Demographic and clinical data for these dogs were collected.Demographic data included age, breed, sex and neuter status.Clinical data included the results of: (i) clinical examination; (ii) abdominal ultrasonography; (iii) thoracic radiography in two projections (lateral and dorso-ventral); (iv) complete blood count including total and differential white blood cell count, red blood cell count, haemoglobin concentration, haematocrit and platelet count; (v) blood biochemistry including the concentration of total protein, albumin, total bilirubin, creatinine, urea and total calcium (tCa) corrected for albumin con-centration (Ca corr ) according to the following formula: Ca corr [mmol/L] = tCa [mmol/L] + 0.02 × (40 -albumin [g/L] (Payne et al., 1973), as well as the activity of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase; (vi) the information about the treatment protocol applied; and (vii) the information about the outcome.

Lymphoma diagnosis
Definitive The morphotype (high-and low-grade) and subtype of TCL were determined based on the updated Kiel classification adapted for dogs as previously described (Fournel-Fleury et al., 1997;Fournel-Fleury et al., 2002;Ponce et al., 2010).The following morphological features of cells were evaluated: size and shape of cells, volume and intensity of cytoplasm staining, size and shape of nuclei, the position of nucleus in a cell; size, distinctness, number and positioning of nucleoli and appearance of nuclear chromatin.Additionally, the mitotic rate in cytological specimens was established according to Meuten et al. (2015).HGTCL was recognised if at least 80% of medium and/or large cells and moderate or high mitotic count (MC) were observed.MC was estimated in cytological smears by using an ocular FN 22 mm (most common) the area of a 'high power (40×) field' = 0.237 mm 2 .A low MC was defined as 0-2 mitosis/ fields, medium as 3 to 5/fields and high as 6 or more mitoses/fields (Ponce et al., 2003).The HGTCLs were further classified into three morphological subtypes according to the updated Kiel classification: pleomorphic mixed (PM), lymphoblastic lymphoma/acute lymphoblastic leukaemia (LB), and plasmacytoid (P).

Clinical staging
Dogs were staged according to the WHO V-stage criteria for canine lymphomas (Owen, 1980)

Treatment
The dogs were categorised based on the treatment applied into two groups: the palliative therapy (PlT) group in which only supportive or glucocorticosteroid (GCS) therapy was used and the chemotherapy (ChT) group in which chemotherapeutic agents were used.Supportive therapy included non-steroid anti-inflammatory agents and antibiotics if necessary, as well as a special diet.GCS treatment in the PlT group was based on oral prednisolone 1 mg/kg BID.
In the ChT group, chemotherapeutic protocols were divided into basic (COP-cyclophosphamide, vincristine, prednisolone; or CHOPcyclophosphamide, doxorubicin, vincristine, prednisolone; or single chemotherapeutic agents) and modified (CHOP with L-asparaginase and/or lomustine; custom treatment schemes).GCS pre-treatment was defined as any use of GCS (oral or injectable) between FNB and the onset of chemotherapy but exceeding a week's treatment.

Outcome
The outcome was defined as the overall survival time (OS) in the PlT group and as the achievement of complete remission (CR) and OS in the ChT group.OS was the time from the day of FNB based on which the final diagnosis of HGTCL was made through the day of death.No observations were censored since all dogs were followed until death, and all deaths were considered as related to HGTCL.CR was defined as complete resolution of all signs of disease, including normal-size lymph nodes.Partial remission was defined as >30% reduction in the mean longest dimension of lesions (Vail et al., 2010), based on the assessment of the size of lymph nodes registered in the medical records.

Statistical analysis
Numerical variables were presented as the median, interquartile range (IQR) and range and compared between groups using the Mann-Whitney U test (two groups) or Kruskal-Wallis H test (three groups).
Categorical variables were expressed as the count and percentage of cases in a group and compared between groups using the maximum likelihood G test or Fisher exact test, if the expected count in any cell of the contingency table was below 5.The Wilson score method was used to work out 95% confidence interval (CI 95%) for proportions (Altman et al., 2000).The link between the HGTCL subtype and the outcome was analysed according to a two-level procedure separately in the PlT and ChT groups.First, the univariable analysis was carried out using the univariable Cox proportional-hazard model (OS analysis) or simple logistic regression (CR analysis); the magnitude of the effect was reported as the crude hazard ratio (HR crude ) or the crude odds ratio (OR crude ), respectively.As OS was exactly known for all dogs and all deaths were considered as related to HGTCL, there were no censored observations in the survival analysis.If the HGTCL subtype proved significantly linked with the outcome, the multivariable analysis was run including the morphological subtype of HGTCL, and these demographic and clinical variables had p < 0.1 in the univariable analysis.The multivariable analysis was performed using the multivariable Cox proportional-hazard model with a backwards stepwise elimination procedure, and adjusted hazard ratios (HR adj ) were reported.OS was graphically presented using the Kaplan-Meier plots.
All statistical tests were two-sided.A significance level (α) was set at 0.05 except for the univariable analysis in which α = 0.1.The statistical analysis was performed in TIBCO Statistica 13.3 (TIBCO Software Inc.).

Study population
The study population consisted of 58 dogs with HGTCL-30 males (nine neutered) and 28 females (15 spayed), aged 1 to 14 years with a median (IQR) of 7 (6-8) years.Forty-seven dogs (81%) were pedigreed with 18 boxers, followed by Dogue de Bordeaux (six dogs), American  1).According to the WHO classification, the first two subtypes (PM and P) corresponded to the peripheral TCL not otherwise specified, and the latter subtype (LB) corresponded to the precursor TCL/leukaemia.There were no discrepancies in the lymphoma classification between the first and the second examination of slides nor between plain microscopic examination and immunophenotyping.Multicentric lymphoma was present in 52/58 dogs (90%, CI 95%: 79%-95%) and 5/58 dogs (9%, CI 95%: 4%-19%) had the mediastinal form (three PM and two P types; Table 1).
One dog (an 8-year-old spayed female boxer) had also signs of cutaneous involvement (PM subtype) and it was also the dog that survived the longest time (367 days) without chemotherapy, only on GCS.On presentation, clinical signs were observed in 50/58 dogs (86%, CI 95%: 75%-93%), including all dogs with the mediastinal form and one dog with the cutaneous form of HGTCL ( weeks, IQR-1 week to 3 months; p = 0.007; Table 2, Figure 1).Therefore, the prognostic role of the HGTCL subtype was further analysed separately in the PlT and ChT groups ( non-PM dogs in either group (Table 3); however, due to small group sizes, the power of these comparisons was poor, and therefore all variables were included in the univariable OS and CR analyses to avoid missing potential confounding factors.

PlT group
Seventeen out of 23 dogs (74%) had the PM subtype and 6/23 dogs (26%) had the non-PM subtype of HGTCL (five P and one LB); 8/23 dogs (35%) received only supportive therapy, while 15/23 dogs (65%) were treated with GCS.PM subtype and two other variables (GCS treatment and being a neutered female) had p < 0.1 in the univariable survival analysis (Table 4) and were entered into the multivariable survival analysis.Of them PM subtype and GCS treatment proved significantly and independently linked to longer OS (Figure 2) and approximately three-fold lower HR adj , although due to small group size precision of estimations was poor (wide CI 95%; Table 5).

ChT group
Twenty a Presented as the median, interquartile range (IQR) and range in parentheses.
b Only median and range presented due to small number of dogs.
*Included in the multivariable analysis.
univariable analysis also showed that >7 days elapsing between diagnosis and the beginning of chemotherapy and GCS treatment prior to chemotherapy were associated with a significantly lower chance of CR (p = 0.034 for both).Additionally, at least 7 days of GCS treatment prior to chemotherapy was associated with shorter OS (p = 0.016); also chemotherapy based on the modified CHOP protocol was associated with a significantly higher chance of CR (p = 0.034) and longer OS (p = 0.039), and CR was linked to significantly longer OS (p = 0.001) (Tables 4 and 6).

DISCUSSION
Our study provided one obvious and one more sophisticated conclusion.The former is that any chemotherapy significantly extends the OS of dogs with HGTCL, compared to no or sole GCS therapy.This has been clearly evidenced in previous studies (Brown et al., 2018;Brodsky et al., 2009;Morgan et al., 2018;Rebhun et al., 2011).The difference in median OS in our study was three-fold (6 vs. 18 weeks).OS of nontreated dogs was only slightly shorter from commonly accepted figures The Kaplan-Meier survival curves of dogs with HGTCL treated palliatively with pleomorphic mixed (PM) and other subtypes according to Kiel classification.Arrows indicate median overall survival time (OS) for each group.CI 95%, 95% confidence interval; HR adj , adjusted hazard ratio.(Bell et al., 1984;Squire et al., 1973), probably due to some reluctance of Polish dog owners to pursue more sophisticated diagnostic procedures like FNB (if the time that elapsed from the first symptoms through FNB to death is added to OS, it corresponds to figures reported in the literature).What is surprising is a very short OS of dogs treated with chemotherapy in our study and a low proportion of CR achieved (roughly more than one-third of dogs).This applies both to standard COP/CHOP protocol as well as more advanced protocols with L-asparaginase and lomustine, even though the latter dogs survived significantly longer.The poor prognosis of dogs on chemotherapy may be to some extent explained by a considerably delayed beginning of chemotherapy in a high number of dogs and frequent use of GCS before entering chemotherapy.These factors are well known to negatively affect response to chemotherapy (Gavazza et al., 2009;Price et al., 1991), and this negative relationship was also apparent in our study.Nevertheless, it would be naïve not to look for the culprit in the chemotherapy or previous steroid therapy because difficulties must also be taken into account, such as irregular medication administration, or the need to modify drug doses due to increased side effects and poor general condition of dogs.
More important appears to be the second conclusion that the morphologic subtype of HGTCL has prognostic value in dogs, which are not treated with chemotherapy.Independent of the use of GCS, dogs with the PM subtype are expected to survive longer than dogs with the non-PM subtype.This observation applies in fact to the comparison between PM and P subtype as only one non-treated dog had the LB subtype in our study, and its OS was 48 days, which is similar to the median OS of PM dogs.Therefore, our study in fact shows that the P subtype is an unfavourable prognostic in dogs whose owners do not opt for chemotherapy.This result is not surprising as the P subtype appears to be the most aggressive of all HGTCL (Ponce et al., 2003;Sayag et al., 2018).It is important to stress that we would not be able to detect any differences between subtypes if we used the WHO classification as PM and P are grouped there together as peripheral TCL are not otherwise specified (Valli et al., 2010).This observation may have applications in clinical practice.Most of the information available concerns treated dogs, and there are not many references to untreated dogs.
More data concern steroid therapy, which is an obvious truth that it improves the general condition of the animal.Many owners, for various reasons, opt out of chemotherapy.However, they want to obtain information about the course of the disease.Therefore, knowledge about factors that positively influence the prognosis in patients undergoing palliative treatment may prove valuable.
The role of morphological subtype turned out to be insignificant in dogs treated with chemotherapy.Obviously, it can be a false negative result due to the very low power of this study as a single previous study reported significant differences in OS between dogs with three subtypes of HGTCL with P associated with the shortest and PM with the longest OS (Ponce et al., 2004).This study included a similarly low number of dogs to ours (10 PM, 10 LB and eight P), so its results should also be treated with caution.Generally, no strong evidence indicating a prognostic role of HGTCL subtype in dogs undergoing chemotherapy has so far been published (Sayag et al., 2018;Zandvliet, 2016).It is possible that the influence of chemotherapy on the natural course of HGTCL is sufficiently strong to suppress any variations related to the morphologic subtype, which would be apparent if the disease was allowed to follow its natural course.This is in fact what our study implies although it is still only a hypothesis that requires further thorough prospective investigation on a markedly larger study population.
At this time, rarely are non-treated dogs the subjects of scientific studies since most current veterinary research focuses on various chemotherapeutic protocols.However, we think that our observation may have some practical value.Even though thanks to increasing owners' awareness and the popularity of pet health insurance, currently diseased dogs have access to modern therapies (Lloyd, 2006), there will always be situations in which palliative will be chosen as a result of financial constraints or a certain system of values of owners and doctors (Gates et al., 2019).Therefore, the knowledge of factors that positively affect the outcome in palliatively treated patients may turn out to be valuable.
In our study population, we found only three morphological subtypes of HGTCL.This is fewer compared to four (pleomorphic large cell and PM cell [small and large], lymphoblastic and P) described in The highest discrepancy was in the PM subtype as all previous studies identified distinct pleomorphic large cell and PM cell small and large.
We did not observe any significant differences in the demographic characteristics or clinical presentation of dogs.Neither did we find any significant difference in CR or OS between dogs with I-III and IV and V stages.We decided on this division for two reasons: First, some stages were very rare in the compared groups (e.g., only one dog with PM subtype and Stage V); second and more important was that accurate differentiation between clinical stages requires a full panel of diagnostic tests including bone marrow analysis as well as more sensitive imaging diagnostic tests than radiographs and ultrasonography applied also to the central nervous system.These tests were unavailable in most of our patients, so it is quite likely that many of them would have been reclassified to a higher stage if additional diagnostics were performed (Vail et al., 2010).This especially applies to the reclassification from Stages IV to V, which is crucial as Stage V is in fact the one whose prognostic role has so far been implied (Zandvliet, 2016).Most studies to date have not found a link between clinical stages and prognosis (Bennett et al., 2023;Dobson et al., 2001;Garrett et al., 2002;Greenlee et al., 1990;Hahn et al., 1992;Keller et al., 1993;Kiupel et al., 1999;Valerius et al., 1997).Some studies found a negative prognostic role of the 'b' substage (Keller et al., 1993;Valerius et al., 1997), but almost all dogs in our study were ill, so we could not include this factor as a potential confounder.
Using GCS in dogs with HGTCL turned out to be significantly associated with longer OS, which agrees with current knowledge and observations in all canine lymphomas irrespective of the immuneor morphotype (Zandvliet, 2016).Given that GCS treatment usually improves the quality of a dog's life, its use is highly recommended in dogs whose owners do not decide on chemotherapy.It is crucial, however, to make the owners aware of the negative effect GCS treatment has on further chemotherapy if they change their mind later.Some of the dogs enrolled in our study had already been treated with GCS by a general practitioner before making a tentative diagnosis of lymphoma and referring the dog to an oncologist.Regardless of the scenario, GCS treatment prior to chemotherapy should be considered as a negative prognostic factor.
In conclusion, our study implies that the morphological subtype of HGTCL has some prognostic value in dogs treated palliatively (with the PM subtype associated with longer OS than the P subtype); however, this effect is no longer visible when a dog is treated with chemotherapy.
diagnosis of HGTCL was based on cytology followed by immunocytochemistry.Smears for cytological examination were obtained by a fine-needle aspiration or non-aspiration biopsy (FNB) from enlarged lymph nodes (in all dogs; in cases of systemic lymphadenomegaly, at least three samples were collected from at least two enlarged lymph nodes) and from additional sites, including enlarged internal organs (splenomegaly in 40 dogs), abnormal masses located on the skin (in one dog) and in the body cavities (mediastinal tumours in five dogs), bone marrow with atypical cells in peripheral blood in five dogs, depending on the disease presentation.For routine examination, at least three smears of aspirates were used.The smears were dried, fixed in 70% methanol, stained with Giemsa solution, and examined by light microscopy (Olympus CX21).The immunophenotype of lymphoma (B cell or T cell) was determined using immunocytochemistry.Briefly, smears from each dog were dried, fixed in acetone at 4 • C for 5-10 min and stained immediately or stored at −20 • C. Immunocytochemical staining was performed according toCaniatti et al. (1996) and Sapierzyński (2010) using commercially available antibodies (Dako) for the pan-T-lymphocyte marker CD3 (polyclonal rabbit anti-human) and B-cell antigen receptor complex CD79α (monoclonal mouse anti-human).Two smears from the same case were stained using both antibodies.The intensity of expression of examined CD antigens in cytological preparations was determined in the light microscope, and the result was considered positive if at least 80% of large or medium-sized lymphocytes showed a strong cytoplasmic reaction.CD3 and CD79α immunopositive cells were counted from 1000 cells using an Olympus BX41 microscope coupled to a computer equipped with a CellA analysis system (images of microscopic fields abundant in well-preserved cells were captured and formatted as TIFF files, and immune expression of CD3 and CD79α was calculated).A TCL immunophenotype was identified if at least 80% of lymphomatous cells were CD3-immunopositive.Negative controls were processed in the same manner, using a buffer solution instead of primary antibodies.The positive controls for CD3 and CD79α were cellular samples collected from impression smears of canine hyperplastic lymph node.Immunocytochemistry results were interpreted without information from routine cytology.
Demographic and clinical characteristics of dogs with high-grade T-cell lymphoma (HGTCL) enrolled in the study with division into two treatment groups.The multivariable Cox proportional hazard model showing the effect of chemotherapy on the risk of death of high-grade T-cell lymphoma (HGTCL) controlled for clinical stage and subtype according to updated Kiel classification.

Table 3
). Demographic and clinical characteristics of dogs did not differ significantly between PM and

PlT group (n = 23) ChT group (n = 35) Subtype according to Kiel classification Variable PM (n = 17) Non-PM (n = 6) p-value PM (n = 27) Non-PM (n = 8) p-value
The relationship between demographic and medical characteristics of dogs in the PlT and ChT group and their OS times.
The multivariable Cox proportional hazard model showing the effect of variables on the risk of death of HGTCL in the PlT group.The relationship between demographic and medical categorical characteristics of dogs in chemotherapy group and the occurrence of CR.